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本研究旨在克隆小鼠adam10基因启动子,构建以adam10基因启动子为启动序列的双荧光素酶报告基因系统并分析其活性,为研究adam10基因转录调控提供有效工具。以BALB/c小鼠脑组织为模板,采用PCR方法克隆小鼠adam10基因启动子序列至pCR-Blunt载体,酶切后连接至荧光素酶报告质粒pGL4.10启动子区域,构建重组荧光素酶报告质粒pGL4.10-adam10,采用阳离子脂质体法与阳性对照质粒pGL4.74共转染真核细胞293FT,以离子霉素刺激为刺激组、DMSO为未加干预组,同时以不含启动子的pGL4.10与阳性对照质粒pGL4.74共转染组为阴性对照组,化学发光仪检测荧光强度及比例。结果表明,酶切及测序验证克隆的adam10启动子序列正确且无突变,构建的重组荧光素酶报告质粒pGL4.10-adam10与阳性对照质粒pGL4.74载体成功共转染293FT细胞,pGL4.10-adam10转染细胞后具有转录活性。离子霉素刺激组活性明显高于DMSO组,荧光比值约为DMSO组2倍(P<0.05),阴性对照组不具备转录活性。结论:成功克隆了adam10启动子并构建了重组荧光素酶报告质粒pGL4.10-adam10,离子霉素可增强adam10基因启动子转录活性。
This study aimed to clone the promoter of mouse adam10 gene and construct the dual luciferase reporter gene system with the promoter of adam10 as a promoter and analyze the activity of the gene. This study provides an effective tool for the study of transcriptional regulation of adam10 gene. The mouse adam10 gene promoter sequence was cloned into the pCR-Blunt vector using the brain tissue of BALB / c mice as a template, and ligated into the luciferase reporter plasmid pGL4.10 promoter region to construct a recombinant luciferase Reported plasmid pGL4.10-adam10, using cationic liposome method and positive control plasmid pGL4.74 co-transfected eukaryotic cells 293FT ionomycin stimulation stimulated group, DMSO was not intervention group, at the same time with no start The sub-pGL4.10 and the positive control plasmid pGL4.74 co-transfection group as a negative control group, chemiluminescence instrument to detect the fluorescence intensity and the ratio. The results showed that the cloned adam10 promoter was correctly and without mutation. The recombinant luciferase reporter plasmid pGL4.10-adam10 and the positive control plasmid pGL4.74 vector were successfully co-transfected into 293FT cells, pGL4.10 -adam10 transfected cells with transcriptional activity. The activity of ionomycin stimulation group was significantly higher than DMSO group, the fluorescence ratio was about 2 times DMSO group (P <0.05), negative control group does not have transcriptional activity. CONCLUSION: The adam10 promoter was successfully cloned and the recombinant luciferase reporter plasmid pGL4.10-adam10 was constructed. Ionomycin can enhance the transcriptional activity of adam10 promoter.